Amidohydrolases (amidases) catalyze in vivo the hydrolysis of amide bonds to the corresponding amine and carboxylic acid. The catalytic properties of amidases are of great interest to develop new drugs and/or pharmaceutical intermediates with cytotoxic activity. While these amidohydrolases have potential for industrial applications, few have been described to date, and they are limited by substrate specificity and stability problems. The application of microbial enzymes for organic synthetic reactions can offer high substrate specificity, complete substrate conversion under mild reaction conditions, and less chemical waste generation. Thermostable enzymes in general offer all these features as well as enhanced stability. ThermoGen proposes to develop a toolbox of stable amidohydrolases and an efficient, scaleable technology for use in organic synthesis reactions. In Phase I we will show feasibility by identifying a set of ten enzymes made up of either new amidases or mutagenized amidases with broader substrate specificity and stability. In Phase II we will develop methods for optimal synthesis conditions, enzyme production, and expansion of the substrate range and further engineer the enzymes. In Phase III, we will commercialize the enzymes and technology for use by synthetic organic chemists and pharmaceutical manufacturers. PROPOSED COMMERCIAL APPLICATIONS: ThermoGen proposes to develop novel amidohydrolases for use as synthetic tools in regio- chemo- and stereoselective organic synthetic reactions. Amidases will help synthesize anticancer drugs or intermediates efficiently and economically and will reduce the chemical waste generation.